Compositions and Methods for the Prevention and Treatment of Metabolic Diseases

ABSTRACT

The present invention is a new method and composition that enhance bone repair, formation, maintenance and slowing of bone resorption. The present invention relates to methods and compositions that enhance collagen formation, tendon health and tendon injury healing, bone maintenance and bone injury healing, and the prevention and treatment of metabolic diseases. The present invention is a part of the therapy to maintain bone health among patients with diabetic bone resorption and others with metabolic disorders. In one embodiment, the composition is a composite of hydroxyapatite and an organic matrix composed of milk pH-dependent serum proteins, i.e., bone morphogenic proteins (BMP), milk serum-derived specific proteins (MSSP), and milk serum derived proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/050,810 filed Oct. 10, 2013, which is acontinuation application of U.S. patent application Ser. No. 13/567,727filed Aug. 6, 2012, now U.S. Pat. No. 8,557,296, which is a continuationapplication of U.S. patent application Ser. No. 12/890,090 filed Sep.24, 2010, now U.S. Pat. No. 8,518,453, which claims priority to U.S.Provisional Application Ser. No. 61/245,505 filed Sep. 24, 2009. Thedisclosure of each application is incorporated by reference herein inits entirety.

FIELD OF THE INVENTION

Bone is an active body system, which tends toward an equilibrium betweenbone formation (osteoblastic activity) and bone resorption, or loss(osteoclastic activity). Normal bone is composed of 65% mineral matrix,primarily composed of calcium hydroxy apatite and other minerals, therest comprising organic or protein matrix materials. Ninety percent(90%) is made of collagen Type 1 and the remaining 10% is composed ofnon-collagenous proteins comprising calcium binding proteins, adhesiveproteins and mineralizing proteins consisting of enzymes, cytokines andgrowth factors. Osteoporosis is a condition where there is a shifttoward increased bone resorption, resulting in a net bone loss, makingthe bone more fragile and prone to breakage.

Osteoporosis, with its precursor condition of bone loss, is a serioushealthcare issue that affects 44 million Americans. Many more, includingyoung persons, are at risk of osteoporosis (1-4). Recent research intothe biochemistry of bone formation and the suppression of boneresorption has led to the development of new therapies and programs forthe prevention and treatment of bone loss and osteoporosis. Milkcontains the best-known sources of nutrients for healthy bone growth inyoung persons and maintenance of bone in the adult populations. Inaddition to milk-based calcium, milk contains a balanced bio-availablemineral profile, bone morphogenic proteins (BMP), milk serumcarbohydrates and essential fatty acids for the growth, development andmaintenance of bones, teeth and skeletal structures. Milk derived basicproteins (MBP) have been shown to promote bone formation and slow excessbone resorption.

Hydroxyapatite (HAP), particularly calcium hydroxyapatite, is the majorconstituent of mammalian bone. It is a derivation of apatite class (themost phosphorus-bearing materials) with isomorphous series:

Ca₁₀(PO₄)₆(CI,F,OH)₂:chloro, fluoro and hydroxyapatite.

Various studies in recent years have focused on HAP growth mechanismsfrom extra-cellular body fluids (human blood plasma).

Proteins, specifically acidic or basic proteins, most likely play animportant role in nucleation and growth modification, similar to otherbio-mineralization systems such as biogenic CaCO₃. It is believed thatpeptides are instrumental in inhibiting growth of HAP in a particulardirection or surface planes (crystal faces). Specifically, the roles of,for example, glutamate (R—COO⁻) versus phosphorserine (R-PO₄ ²⁻) aminoacid residues is highly controversial and is under extensive researchscrutiny.

Milk, specifically domesticated cow milk, is a primary source of thespecies and nutrients discussed above. Milk provides nourishment andimmunological protection for mammalian young and is a source of food,minerals and other nutrients for more mature mammals. Milk is a verycomplex food containing, in one estimation, over 100,000 molecularspecies.

BRIEF SUMMARY OF THE INVENTION

Compounds and formulations of this invention are a milk-basedtherapeutic formulation, which comprise milk serum-specific proteinswhich are basic or acidic in nature, bone morphogenic proteins and otherfactors capable of promoting bone formation and inhibiting boneresorption. The examples and disclosure herein demonstrate the efficacyof this invention in slowing bone resorption and provide evidenceinferring compounds of this invention actually stimulate bone formation.

Briefly, in one aspect, the present invention is a new product forcollagen-formation, collagen repair, bone repair, bone formation,maintenance, and regeneration. This invention is a nano-composite ofhydroxyapatite nano-fibers and an organic matrix composed of milkpH-dependent serum proteins, i.e., bone morphogenic proteins (BMP), milkserum-derived specific proteins (MSSP), milk serum derived proteins,both basic and acidic in nature with various positive and negativecharges. For purposes of this invention, “milk serum proteins” aredefined as milk minus naturally occurring casein or casein-free milk.Representative serum proteins present in this invention include but arenot limited to β-lactoglobulin (formerly called lactalbumin),α-lactoglobulin (formerly called lactalbumin), Lactoferrin,Lactoperoxidase, IgG, IgM (‘m’ chain), secretory piece, (secretory pieceis a glycoprotein often found associated with IgA), α-casein, bovineserum albumin, IgA (‘a’ chain) or IgD (‘d’ chain), glycoproteins, caseinphosphopeptides, lipoprotein A1, retinol-binding protein, osteopontinand its fragments and other minor proteins such as growth Factors andpre-albumin. It exists as hydroxy-apatite in natural form and thereforeis hexagonal in shape.

Applicant has developed, and discloses herein, a new milk-basedtherapeutic formulation containing for example, the patented material,of U.S. Pat. No. 5,639,501, sometimes referred to herein as “DariCal”,containing a unique combination of two forms of high quality milkcalcium—calcium phosphate and calcium lactate. The DariCal materialincludes minerals with high bio-availability, Proteins, carbohydratesand fatty acids. A compound of the current invention, herein sometimereferred to as “Hexaminacol” for identification purposes in addition tothe above, contains milk-serum-derived specific proteins of acidic andbasic in nature (MSSP) and bone morphogenic proteins (BMP) such asalpha-lactalbumin, beta-lactglobulin, immuno-gammaglobulin, growthfactors, Lactoferrin, Lactoperoxidase and other valuable peptides andamino acids. These proteins, peptides and amino acids have been shown tostimulate bone growth and slow down bone resorption. The Hexaminacolmaterial has approximately the same mineral profile (proportions andcontent) as bone (Table 1) and provides these organic precursors andinorganic components necessary for the manufacture and maintenance ofbone.

TABLE 1 Hexaminacol ™ Mineral Analysis Compared with Bone ElementHexaminacol Bone Calcium (Ca) 26.0% 25.0% Phosphorus (P) 15.5% 12.0%Magnesium (Mg) 1.5% 0.37% Potassium (K) 0.5% 0.5% Zinc (Zn) 15.0 ppm 9.0ppm Iron (Fe) 26.0 ppm 21.0 ppm  Manganese (Mn)  1.5 ppm 1.2 ppm Copper(Cu)  3.2 ppm 0.5 ppmIn addition to the mineral analysis the other materials present in thecompound are: the total protein content in Kjeldahl analysis may rangefrom 2.0% to 10% depending on the need to maintain the protein contentand type of proteins desired for a particular function. The product mayalso contain fatty acid content between 0.04% to 0.06% with a fatty acidprofiles of cupric acid, caproic acid, Linoleic acid and oleic acidgroups. The product may also contain total carbohydrate (CHO) content inthe range of 4 to 9% composed of lactose, glucose, maltose and fructose.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be illustrated by the attached Figures, DetailedDescription and appended Claims, all of which should be consideredexemplary and not limiting of the present invention and in which:

FIG. 1 is a flowchart showing one embodiment of method used in thisinvention;

FIG. 2 is a TEM image showing aggregates of hydroxyapatite nano-fibersof the new invention;

FIG. 3 are TEM images showing bundles and aggregates of hydroxyapatitenano-fibers of the new compound. The amorphous organic “matrix” can beclearly seen along the thin edge areas. Both images were recorded undersame magnification;

FIG. 4 is a high-resolution TEM image showing lattice fringes(crystalline nature) of the nano-fibers crisscrossing like a woven cloth(overlap of many crystals) as compared to calcium carbonate and calciumhydroxyapatite, which run in a single direction. As shown thelattices-overlap and cross to form layer upon layer of crossing fibrils.

FIG. 5 is a TEM image of the new compound showing the amorphousCI-bearing Ca-phosphate aggregates in solution at pH=2.0;

FIG. 6 is an XRD pattern for the new compound sample;

FIG. 7 is an X-ray diffraction pattern and simulated pattern (best fit)for the new compound apatite;

FIG. 8 is a 3-D model showing atoms within the unit cell. Red: oxygen;Blue: OH (basically shows O of OH); Blue/green: Ca; Purple-brown: P;

FIG. 9 is a 3-D polyhedral model of apatite structure. P are intetrahedra (Purple-brown color). Ca are in poly hedra with coordinationnumbers of 7 and 9 respectively;

FIG. 10 is a projection of the atoms within a ½ unit cell along c-axis(or on x-y plane);

FIG. 11 is a polyhedral model of the apatite structure projected alongc-axis (or, on x-y plane). P are in tetrahedra (light brown color). Caare in polyhedra with coordination numbers of 7 and 9 respectively;

FIG. 12 is a projection of the atoms within unit cell along [100]direction (or on y-z-plane); and FIG. 13 is a polyhedral modelschematically showing an apatite nano-fiber of the invention.

FIG. 14 is a photomicrograph of a product of this invention such as itappears after drying step (e.g., spray drying) 16 in FIG. 1 (scale isshown).

FIG. 15 is a photomicrograph of the material of FIG. 14 at highermagnification (scale is shown).

FIG. 16 is a comparison of current therapies, the XRD pattern commercialCalcium Carbonate, USP Pharma grade supplier Generichem Corp. commonlyused in the manufacture of calcium tablets. Surface Area: generallyabout 2.7 m²/g. XRD patterns from the sample (upper plot) and peaks fromreference file in data base (lower plot). The diffraction patternmatches calcite very well. This FIG. is included for purposes ofcomparison.

FIG. 17 is the XRD pattern of synthetic calcium hydroxyapatite fromAldrich chemicals, St. Louis, Mo. Surface Area: 29.6 m²/g. XRD patternsfrom the sample (top plot) and peaks from reference files in data base(middle and lower plots). The diffraction pattern matches hydroxyapatitevery well. Relatively strong intensities and sharp peaks indicate largeapatite crystals.

FIG. 18 is the XRD pattern of Hexaminacol a compound of this invention.The vertical lines indicate the peak positions of hydroxyapatite fromthe PDF2 database provided by ICDD.

FIGS. 19 and 20 is a low-magnification TEM (transmission electronmicroscope) image showing calcium carbonate crystals and theiraggregates. Crystal size ranges from 200 nm to 2 microns in a singledirection.

FIGS. 21 and 22 show high magnification TEM images showing latticefringes in a calcium carbonate crystal, formation in a single direction.

FIGS. 23 and 24 are low-magnification TEM images of synthetic hydroxyapatite crystals and their aggregates (synthetic hydroxy from AldrichChemical, St. Louis, Mo. U.S.A.). Crystal size ranges from 50 nm to 100nm.

FIGS. 25 and 26 are high magnification TEM images of synthetic calciumhydroxyapatite from Aldrich chemicals showing lattice fringes in theapatite crystals in a single direction.

FIG. 27 shows a detailed macrostructure and microstructure of humanvertebra showing the relative average sizes of the indicated structures.Hexaminacol resembles the nanostructure of the bone when rolled.

FIG. 28 shows the rate of change for nmBCE as compared to age for theparticipants in Table 3.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to FIG. 1 there is shown a flow chart of a process of thepresent invention. As is shown, at box 1 there is a raw material inputof treated whey from some “other” whey treatment process meaning atreatment process other than one of this invention. An exemplary wheytreatment process, as was noted above, is described in U.S. Pat. No.5,639,501 to Raj an Vembu et al., the teachings of which areincorporated by reference herein. As is shown in box 1, the previouslytreated whey is decanted into, for example, centrifuge 3 for separationof solids (to box 5) from liquids (to box 7). Other processes forseparation of whey solids from liquids, i.e., other than centrifugation,may be used and are within the contemplation of this invention.

The liquid permeate or supernatant generated in the separation step at 3is transferred in box 7 comprises water, lactose, sodium-based minerals,and soluble polypeptides. As shown, the supernatant of box 7 is exposedto a chromatographic ion exchange process in box 8. Optionally andsequentially, and not normally concurrently, additional skim milk, whey,or whey permeate, whey serum protein (shown in box 9 as being added skimmilk, whey, whey permeate, or liquid whey protein concentrate) is alsosubjected to ionic exchange. The ionic exchange material used in box 8is positively (+) charged, thereby causing negatively (−) chargedproteins to be separated from the liquid by being retained by thecolumn. Positively charged proteins, regardless of source (i.e.,regardless of whether they originated from separation at 3 or were addedas shown in box 9) pass through the positively charged ion exchangecolumn without being retained and proceed to a dialysis step, asnecessary as shown in box 10. The dialysis step as shown in box 10removes substantially all sodium-based milk minerals and sodiumchloride, generating an effluent stream of substantially pure negativelycharged milk serum protein.

Parallel to the process steps shown in boxes 3,7,8,10, and 12 the solidsseparated at 3 are transferred (box 5) and purified 6, e.g., by dilutingthe solids in water and heating the solution to a temperature of atleast about 165° F. to 185° F. for a time period of not more than about1 hour. The solids purified in this step substantially comprise divalentmilk minerals, preferably of the alkaline earth family e.g., Ca²⁺ andMg²⁺ but including other divalent species such as Cu²⁺, and Mn²⁺. Thedivalent milk minerals purified as shown in box 6 are concentrated inbox 14 e.g., by centrifugation. The milk serum proteins originating frombox 12 and the divalent milk minerals originating from box 14 then arecombined (not shown by a separate box) and dried as shown in box 16. Thecrystalline morphology, x-ray diffraction characteristics, elementalcomposition, and surface area of this milk serum protein/mineralcomposite material 18 are described in detail below.

Transmission electron microscopic (TEM) images show aggregates andbundles of the hydroxyapatite nano-fibers with non-crystalline organicsamong them (FIGS. 2, 3). The average diameter of the apatite is about 5nm. The length of the nano-fibers is about tens to several hundreds ofnanometers. The elongation direction is c-axis of the apatite crystal.Non-crystalline foil-like organic materials are “matrix” of the samples.The apatite nano-fibers are “glued” together by the organic materials.The apatite nano-fibers are very reactive and unstable under electronbeam (with respect to normal synthetic apatite crystals).

Description: A compound of the invention exists, in part, ashydroxyapatite in its natural form and therefore is hexagonal in shape.

A compound of this invention promotes bone repair, formation,maintenance, regeneration and collagen formation. The present inventionrelates to methods and compositions that enhance collagen formation,tendon health and healing, bone health and healing, and maintaining andaiding in diabetic bone resorption. The present invention is useful inthe treatment of metabolic disorders. It is a nano-composite ofhydroxyapatite nano-fibers and organic matrix composed of milk serumspecific proteins of basic and acidic in nature with various positiveand negative charges. The proteins are pH dependent and include but arenot limited to α-lacto globulin (formerly called lactalbumin), β-lactoglobulin (formerly called lactalbumin), Lactoferrin, Lactoperoxidase,IgG, IgM (m chain), secretory piece*, (*secretory piece is aglycoprotein often found associated with IgA), {acute over(α)}-(alpha)casein, bovine serum albumin, IgA (a chain) or IgD (dchain), glycoproteins, casein phosphopeptides, lipoprotein A1,retinol-binding protein and other minor proteins such as bovine growthhormone and pre-albumin.

Transmission electron microscopic (TEM) images of a material of thisinvention show aggregates and bundles of the hydroxyapatite nano-fiberswith non-crystalline organics among them (See, e.g., FIGS. 2, 3). Theaverage diameter of the apatite is about 5 nm. The length of thenano-fibers is about tens to several hundreds of nanometers. Theelongation direction is c-axis of the apatite crystal. Non-crystallinefoil-like organic materials are “matrix” of the samples. The apatitenano-fibers are “glued” together by the organic materials. The apatitenano-fibers are very reactive and tend to be unstable under electronbeam exposure with respect to normal synthetic apatite crystals.

While not wishing to be bound by any theories Markus Buehler of theMassachusetts Institute of Technology has opined that unlikeconventional building materials, which tend to be homogenous, bone isheterogeneous living tissue with cells constantly changing. Scientistsclassify bone's basic structure into a hierarchy of seven levels ofincreasing size. Level 1 bone consists of chalk-like hydroxyapatite andcollagen fibrils, which are strands of tough proteins. Level 2 comprisesa merging of these two into mineralized collagen fibrils that are muchstronger than the collagen fibrils alone. The hierarchical structurecontinues in this way through increasingly larger combinations of thetwo basic materials until level 7, or whole bone comprising generally of35% protein or organic matrix and 65% of mineral matrix.

At the molecular level, mineralized collagen fibrils are made up ofstrings of alternating collagen molecules and consistently sizedhydroxyapatite crystals. These strings are “stacked” together butstaggered so that crystals resemble stairs. Weak bonds form between thecrystals and molecules in and between strings.

Pressing the fabric-like fibrils breaks some weak bonds between thecollagen molecules and crystals, creating small gaps or stretched areasin the fibrils. Stretching spreads the pressure over a broader area, andin effect, protects other, stronger bonds within the collagen molecule,which might break outright if the pressure was focused on them.Stretching also lets the crystals move in response to the force, ratherthan shatter, which would be the likely response of a larger crystal. Itis theorized that this ability to absorb energy may reduce bonebreakage, e.g., in a fall.

Buehler observed that bone has a unique ability to tolerate gaps in thestretched fibril fabric. These gaps are of the same magnitude—severalhundred micrometers—as the basic multicellular units associated withbone's remodeling. The units are a combination of cells that worktogether like a small boring worm that eats away old bone at one end andreplaces it at the other, forming small crack-like cavities in betweenas it moves through the tissue.

Thus, the mechanism responsible for bone's strength at the molecularscale also explains how bone can remain strong, even though it containsthe many tiny cracks required for its renewal. Bone creates strength bytaking advantage of the gaps, which are made possible by the material'shierarchical structure. Nov. 1, 2007, “How Bone is Built May Lead to NewMaterials,” http://medicaldesign.com/materials/bone. Visited Mar. 31,2009.

Surface area of the new compound is 207.5 m²/g. It is very high comparedto those of synthetic hydroxyapatite (from Aldrich Chemicals, St. Louis,Mo.) (29.6 m²/g) and a commercial calcium Carbonate product (Pharmagrade powder from Generichem Corporation, NJ used for calcium supplementtablet making) (2.7 m²/g). It can be inferred that the product of thisinvention is very reactive considering its large reactive surface.Preliminary studies show a compound of this invention to be about 76times more reactive than calcium carbonate, a widely used supplement inthe industry.

Chemical Reaction in pH 2 Solutions:

The solution was adjusted by using HCl (0.1M) acid. The amount of 0.05 gof the sample can be dissolved completely in the 100 ml pH2 solution.The final solution is clear with pH value of 3.5. Adding 0.15 g of thesample into the 100 ml pH 2 solution, the final solution is not clear,is opaque and it looks like suspension that contains colloid-likeparticles. The solution of the pH rises to 4.7. The particles in thesuspension were analyzed using transmission electron microscope. Theparticles are amorphous Cl-bearing Ca-phosphate. It is proposed that theoriginal hydroxyapatite nano-fibers were dissolved and the amorphousCl-bearing phosphate can precipitate from the Cl-bearing solution.

Dissolution Kinetics Based on One Set of Dissolution Experiments:

Experimental condition: 0.05 g of Hexaminacol added into 20 ml pH2solutions, and reacted with compound of the invention at roomtemperature for 0.5 hr, 1 hr, 2 hr, and 3 hr. The reaction products werecentrifuged right after they reached the set up reaction time in orderto separate solution and remaining solids. The remaining solids weredried and weighted carefully in order to evaluate the mineralelectrolytes, which are charged and thereby combine with positively andnegatively charged proteins. For example, Ca²⁺ molecules have a positivecharge, and therefore interact and attract negatively charged molecules.

Chemical Formula Based on Chemical Analysis:

The chemical formula is based on the provided results and normalized to3 P. (Ca_(4.963), Mg_(0.0365), Sr_(0.0005))(PO₄)₃ (OH_(0.82), Cl_(0.15),F_(0.03)). Generally, Ca₅ (PO₄)₃.OH with randomly placed Na, K. Themolecular weight is: 506.34 (g per formula).

It is a hydroxyapatite. Na and K are in soluble salt forms. It is alsopossible that trace amount of Mg in soluble form.

The calculated density of the apatite based on measure unit cellparameters and obtained chemical formula is 3.15 g/cm³. In general, itis slightly lower than the calculated value of macroscopichydroxyapatite apatite crystals.

X-Ray Diffraction Analysis:

X-ray powder diffraction pattern shows that the crystalline phase inbulk powder sample is a nano-crystalline apatite (hydroxyapatite). Allthe diffraction peaks are very broad except for a strong and relativelysharp 002 diffraction peak (d=3.416 A). The shapes of the diffractionpeaks indicate that the hydroxyapatite crystals are nano-fibrouscrystals with elongation direction along c-axis.

Unit Cell Refinement:

The unit cell parameters of the New Compound apatite were calculatedbased on a whole pattern refinement method (Rietveld method) based on apublished average structure of hydroxyapatite. The results show thata-dimension and b-dimension of the unit cell are slightly larger thanthat of standard hydroxyapatite. However, its c-dimension is slightlysmaller than that of standard hydroxyapatite. It is proposed thatnano-fiber affects structural relaxation of the apatite structure,especially atoms on surface and nearby surface. It is expected atomiccoordinates for the New Compound apatite structure may be slightlydifferent from the reference structure. However, it is impractical torefine the coordinates based on diffraction pattern with very broaddiffraction peaks.

TABLE 2 Fractional Co-ordinates of the atoms Atom x y z occupancy Ca-10.33333 0.66667 0.0015 1.00000 P 0.3987 0.3685 0.2500 1.00000 OH 0.000000.00000 0.1950 0.50000 O-1 0.3284 0.4848 0.25000 1.00000 O-2 0.58730.4651 0.25000 1.00000 O-3 0.3437 0.2579 0.0702 1.00000

Unit Cell Parameters for the Apatite Compound of this Invention:

a=b=9.500 Å

c=6.821 Å

alpha=90.00°

beta=90.00°

gamma=120.00°

Symmetry: space group P6₃/m.

Unit Cell Parameters of a Reference Hydroxyapatite:

a=b=9.416 Å

c=6.875 Å

alpha=90.00°

beta=90.00°

gamma=120.00°

Symmetry: space group P6₃/m.

Photomicrographs of this material are shown in the Figures.

Subjects and Methods Generally

Subjects.

Participants were menopausal and postmenopausal women recruited from thepopulation at large in the Madison, Wis. area who were otherwisehealthy, not taking any diet supplements and living normal lives.Seventeen women initially signed the consent forms for the study butfour were disqualified for non-compliance of the protocol. Thirteenwomen with ages ranging from forty to seventy-one completed thetwelve-week study. The study protocol complied with HIPAA regulations.

Protocol.

After enrollment, each participant was given a urine NTx assay kit withinstructions to collect the second void of morning urine sample(pre-treatment) and mail the sample with the return overnight mail tothe central testing laboratory (Madison Pharmacy Associates, Madison,Wis.) to establish baseline NTx readings. Participants were notified oftheir initial readings. Normal NTx readings are below thirty-eightnMBCE, an elevated NTx is between forty to sixty nMBCE and a high NTx isabove sixty nMBCE. An elevated NTx is indicative of osteopenia while ahigh NTx is inferred as osteoporosis in correlating to low BMD and boneloss. Selected participants, with NTx readings higher than thirty eightwere mailed Hexaminacol compound in powder form individually packaged intwo gram packets for a twelve week supply along with use instructions,daily intake recording calendars, second test NTx kits and othernecessary contact information. Participants were required to consume twograms of Hexaminacol powder (500 mg calcium equivalent) two times daily,totaling one thousand mgs of calcium equivalent, two times daily, in themorning and one-half hour before bed-time. Participants had unlimitedaccess to information with study monitors. Participants were contactedevery week to verify that they were in compliance with the studyprotocols, maintaining expected records and assessments were made abouthow well the product was being tolerated. After twelve weeks ofHexaminacol intake, the participants who were verified to have been incompliance with the protocol were requested to send a second urinesample (post-treatment) by overnight mail.

Urine NTx (a Bone Resorption Marker)

Urine NTx is a method for examining cross-linked N-Teleopeptides oftype-I collagen (NTx) that the body excretes in urine when bones arebroken down (21, 22). An increased rate of NTx excretion indicates ahigher rate of osteoclast activity and bone destruction. Unlike bonemineral density (BMD) measurements, which typically detect changes inbone density over years (23), NTx is able to detect changes in bonemetabolism in weeks or months (24). Two possible applications of NTx areto (a) predict bone loss in Peri- and postmenopausal women and to (b)monitor the skeletal response to treatment. While NTx testing does notdirectly determine osteoporosis, it does determine the likelihood ofdecreasing bone density, as measured by conventional bone massmeasurements. The higher the rate of bone resorption as measured by NTx,the greater the rate of bone loss (22,25). Elevated levels of boneresorption (NTx) markers found in urine are associated with higher ratesof bone loss in postmenopausal women (26,27).

Bone resorption markers may also play a role in evaluating the effectsof therapy (28). Current osteoporosis treatments act to decrease boneresorption, which is detectable by changes in NTx studies (21). Usingmarkers, the efficacy of treatment may be determined in a matter ofmonths. These shortened timelines for treatment increase feedbackresponse by comparison to changes in bone density that may not bedetected for one or two years. Experts suggest that demonstrating earlyevidence that the osteoporosis regimen may be working can reinforce apatient's desire to continue therapy, enhancing compliance withtreatment. Many specialists treating osteoporotic patients use boneresorptive markers in assessing the role of high bone turnover inpathogenesis and prognosis, as well as assessing the response toantiresorptive drugs. Failure to detect a decrease in bone markers couldindicate a lack of compliance or efficacy of antiresorptive drugtherapy.

Demographics

TABLE 2a Demographics of Subjects Total number of subjects 13 Number ofpre-menopausal subjects 3 Number of postmenopausal subjects 10 Averageage of subjects 58 years Average pre-treatment NTx of all subjects 49.76nMBCE Average pre-treatment NTx of pre-menopausal 44.33 nMBCE subjectsAverage pre-treatment NTx of postmenopausal 51.40 nMBCE subjects n = 13

Status of Subjects.

During the twelve-week study period, none of the subjects reportedbloating, diarrhea, heartburn, allergy symptoms or adverse reactionsfrom the use of the Hexaminacol™ powder. They all maintained theirnormal health with routine living. None reported to face adverseconditions in life, none of the subjects were involved or reported tohave been in any accident, no allergies of any kind or any treatment forany diagnosis of diseases.

Table 3: NTx reading AND Table 3a: Graphs Participant Age Baseline Final% Change (years) (nMBCE) (nMBCE) (nMBCE) 1. 40 43 37 −13.95 2. 44 46 38−17.39 3. 47 44 38 −13.63 4. 52 45 38 −15.55 5. 57 43 33 −23.25 6. 58 4537 −17.77 7. 59 47 38 −19.14 8. 61 44 37 −15.90 9. 62 46 38 −17.39 10.64 50 38 −24.00 11. 69 60 38 −36.66 12. 70 61 38 −37.70 13. 71 73 38−47.94

It is believed that compositions of this invention will be found usefulin the following applications: prophylactic (i.e., prevention) andtherapeutic (i.e., mitigation) inhibition of types of cancer especiallycolorectal and breast muscle, tendon building and repair, Type IIdiabetes; and osteoporosis;

All mammalian applications e.g., veterinary, are intended. For example,a material of the present invention is believed to be applicable to theinhibition of loss, bone mass building, healing of bone fractures, andrestriction or enhancement (as appropriate) of bone mass densitychanges. For example a compound of this invention may be used to treatbone fractures and to build bone mass in veterinary applications. Forexample, dogs have specific applications such as fracture healing andbone mass strengthening or enhancement. Hip dysplasia in dogs is alsothought to be treatable i.e., to inhibit the onset or to cause thedysplasia to be reduced or healed using the present compound. Thecurrent invention compound, Hexaminacol™ because of its large surfacearea of 207.5 m2/g is believed to be ideally usable as a carrier formedicines or agents, in the manufacture of tablets, capsules and todeliver in a powder form both in bulk and in smaller delivery doses.

Example 1

The focus of this study is to use computational techniques to determineat the atomic level, the mode of interaction between the HAP and______protein/peptide/amino acid factors and milk proteins and todetermine whether glutamate or phosphoserine residues are preferred incontrolling HAP nucleation and crystal growth.

Classical molecular mechanics/molecular dynamics (MM/MD) techniqueimplemented in FORCITE (Accelrys™) code was used. The MM calculationprovides single point energies and geometry optimizations for bothmolecules and periodic systems.

MD simulations were performed for the NVT ensemble at 300 and 500K for 5ps with a 1 fs time-step for BSP in solution. For periodic systems,quench dynamics at 350 K for 5 ps with 1 fs time-step were used to givepreferential adsorption sites on the surface before geometryoptimizations.

Reliability of surface energies and adsorption energies in classical MMsimulation relies on the accuracy of potentials. We chose the UniversalForce Field (UFF) potentials that were derived for organometallics,hence, should handle our systems fairly accurately.

Example 2

Classical molecular modeling techniques were used to examine the mode ofinteraction between specific crystal faces of HAP or aqueous Ca²⁺ andHPO₄ ²⁻ ions and an acidic peptide containing glutamate (R—COO⁻) andprotonated phosphoserine (R—HPO₄ ⁻) amino acid residues. The peptiderepresents the postulated active part of bone sialoprotein (BSP) incontrolling bone HAP nucleation and crystal growth modification.

Our preliminary results presented here, suggests stronger peptideattachment on the (0001) surface than the (1010) surface of HAP for bothglutamate and protonated phosphoserine sites. Further, there is nopreference of either group for adsorption.

Equilibrating peptide in solution shows that HA will possiblypreferentially nucleate at the site containing glutamate groups, with aminimum number of eight glutamate sites required in the peptidestructure for nucleation to take place.

Example 3 Pharmacokinetics and Bioavailability Study

A group of 40 menopausal women subjects (acting as their own control)ranging in age from 40 to 65 were selected. Clinical studies, which wereconducted at the University of Wisconsin Medical School (Madison, Wis.),followed all Institutional Review Board (IRB) protocols and patientconfidentiality procedures. In this study (See table below), acomposition of this invention was compared against OsCal®, an availablecalcium supplement commercially available from Merk. The same group ofmenopausal females functioned as control group in the study. The studywas conducted in two phases, the first one being the control phase usingOsCal and the second phase being where a product of this invention wasadministered. Subjects were required to avoid all dairy products orsupplements for two weeks and to fast overnight prior to commencement ofthe study. Subjects were administered 500 mg of a compound of thisinvention vs. 500 mg of OsCal, with breakfast. Blood samples were drawnat 0, 1, 2, 3, 5, 7 and 9 hour intervals to establish baseline bloodcalcium and minerals levels over a period of time. A compound of thisinvention was found to be absorbed in the blood stream, was determinedsafe to the subjects, with no adverse reactions or detrimental sideeffects. The results showed OsCal subjects had calcium in their bloodserum. However, while invention compound subjects had calcium in theirblood serum, the results showed the presence of mineral nutrients knownto be important to bone formation. This study established the absorptionand safety of a compound of this invention.

OsCal ® Invention p Calcium Cmax (mg/dl) 0.47 ± 0.25  0.43 ± 0.234 0.57Serum calcium Tmax (hr) 3 3 0.87 (2, 5) (1, 5) Mean change in serumCalcium AUC (mg-hr/dl) 1.67 ± 2.32 1.25 ± 1.96 0.54 Phosphate (mg/dl)−0.10 0.40 0.006 (−0.3, 0.15) (0.00, 0.675) Potassium (mEq/L) 0.01 ±0.34 0.20 ± 0.20 0.08 Magnesium (mg/dl) 0.01 ± 0.09 0.04 ± 0.10 0.29Iron (mcg/dl)  3.55 ± 13.40  6.53 ± 14.35 0.52

Example 4 NTx Performance Study (Biomarker for Bone Loss)

Thirteen menopausal women age 50 to 71 completed this bone restorationstudy. NTx is a urinary assay for the measurement of the excretion ofcross-linked N-Teleopeptides of Type I Bone Collagen. Bone formation andresorption is normal with a degree of collagen present in urine fordisposal of collagen waste. The presence of abnormally high levels ofType 1 Collagen in the urine is an indication of bone loss.

All subjects were initially measured for their NTx readings to establishtheir baseline status. A normal NTx is a reading below 38 nanomoleculesof collagen in urine (nMBCE), Elevated NTx levels are; 38 to 60, HighNTx levels are above 60. Normal pre-menopausal women have a reading ofabout 38. Participant subjects' NTx measurements ranged from 43 to 79.The subjects were administered 2 grams of a compound of this inventionin powder form dissolved in at least four ounces of water twice dailyfor ninety days. At the end of the study period, the subjects wereretested for the presence and levels of NTx. Post-study NTx resultsranged from 33 to 38 nMBCE, indicating that 100% of the studyparticipants were normalized to healthy NTx levels. Results from thestudy showed subjects with the highest NTx readings indicating high riskfactors for fractures, benefited the most from the therapy of theinvention. We believe that these results indicate that compounds of thisinvention are effective in significantly reducing loss of Type 1Collagen found in urine.

Example 5 Clinical Practice Study (Bone Mass Development-BMD)

Post-menopausal women recruited from a hospital clinic and a nearbynursing home. These women were screened for Osteoporosis risk factors.The screening was done with the aid of Quantitative Ultra sound (QUS)using Achilles Insight (Lunar GE Medical). Women with a T-Scoremeasurement of minus 2.5 (−2.5) were referred for DXA (Dual X-RayAbsorpotiory) measurement for diagnosis and confirmation.

The Study: One Hundred postmenopausal women were administered with acompound of the invention and screened for improvement in bone health atthe end of one year. Results from the study indicate most (87)participants showed improvement in their QUS reading within one year.The study participants continued the therapy for an additional year andcompleted the study. A preliminary and on-going data analysis indicatesthat most patients have gained bone mass at a rate in the range of about5-6% per year over time of the study 2 years. Some participants haveshown more significant improvements.

Bone resorption or bone breakdown was measured using the urine NTx assay(Osteomark, Princeton, N.J.). NTx levels were assessed before treatment(baseline) and after twelve weeks of treatment (final). The meanpre-menopausal score or NTx bone resorption is 38nMBCE.

Table 3/3a presents the data on bone resorption changes at twelve weekscompared to baseline. Eleven out of thirteen subjects had baseline NTxreadings from 43 to 60 (‘Elevated NTx Group’) and the other two subjectshad baseline readings above 60 (‘High NTx Group’). The subjects in thehigh NTx baseline group were ages seventy and seventy-one respectively,while the elevated NTx baseline group ranged from ages forty tosixty-nine. All thirteen subjects experienced a reduction in NTx markerof bone resorption. Twelve out of thirteen subjects had a final NTxreading of 37 or 38 (pre-menopausal mean), however, one subject had afinal NTx score of 33. Percentage change of baseline for the subjectsranged from −47.94% to −13.63% with the most reduction noted with thesubjects in the high NTx baseline group.

Osteoporosis is a disease involving a pathologic bone remodeling processresulting in a shift toward increased osteoclastic activity anddecreased osteroblastic activity leading to a net bone loss, resultingin an increased risk of fractures. This phenomenon normally occurs inwomen during and following menopause. Such bone resorption is seen withexcretion of NTx in urine and can easily be measured to observe andmodify treatment. In the study, baseline urine NTx measurements above 38nMBCE were indicative of abnormal bone resorption with a subset of thegroup in the ‘High NTx’ category i.e. the group at a relatively higherrisk of fractures.

Bone remodeling is a complex process dictated by both organic andinorganic factors. The deficiency of these factors over a period of timeleads to abnormal bone resorption and eventual bone loss. Hexaminacolcontains both organic and inorganic components, which serve as “rawmaterials” for building and maintaining bone. The inorganic componentsare precursors of calcium hydroxyapatite (see Table 1), and the organiccomponents include milk serum basic proteins (MSBP), bone morphogenicproteins, casein phosphopeptides which are the building blocks for type1 collagen and proteins which also facilitate absorptions,transportation and adhesion for the formation of bone material. Thiscompound also contains carbohydrates, essential fatty acids andvitamins. Several studies have shown that milk basic proteins, MBPpromote bone formation.

The subjects in the study, after treatment with OstiGen for twelveweeks, showed a reduction in urine NTx levels to the premenopausal meanof 38nMBCE, which is an indication that their bone resorption at the endof the study was equivalent to bone formation. Baseline at 38nMBCE30nMBCE/creatinine. One of the subjects had an NTx reading of 33nMBCE, avalue much lower than the pre-menopausal mean. This may be attributed toa phenomenon called ‘bone turnover suppression’. The highest percentagechanges of baseline were seen with the older subjects (ages 69 to 71years) recording a range from −36.66% to −47.94%. This subset with thehighest relative NTx values had the most benefit granted during thisstudy. DXA is the standard for testing ‘bone mineral density’ (BMD) butdoes not measure the quality of the bone. Continued loss of type 1collagen, especially in the elderly and increase of the mineral matrixbecause of calcium supplementation gives normal BMD readings but bonebecomes brittle and shatters in the event of a fall. One of theobjectives of this invention is to maintain the ratio of mineral matrix(65%) and organic matrix (35%) in order to prevent illness, injury andfractures. Quantitative Ultra-Sound (QUS) provides not only the BMDreadings but also the quality of the bone mass. In addition to QUS theNTx testing and Bone Alkaline Phosphatase (BAP) testing provide a box oftools in the disease management of Osteoporosis in addition to DXA.

Direct measurement of the rate of bone loss in a patient withosteoporosis would require at least two measurements of bone mass over a2-4 year interval. Such a strategy is not practical where it isnecessary to decide whether or not to treat at the time of initialassessment. Since the rate of bone loss is proportional to the rate ofbone turnover in postmenopausal women, it has been suggested that therate of loss can be predicted by assessing bone turnover that arespecific for bone resorption. Although rates of loss assessed in thismanner are less accurate than rates of loss assessed over many years bysequential bone mineral density (BMD) measurements, a high rate of boneresorption above the premenopausal mean is associated with a 2-foldincrease risk of vertebral and hip fracture independently of theprevailing BMD. The study revealed the resolution of abnormal boneresorption to premenopausal levels with the use of Hexaminacol,indicating that the presumed on-going bone resorption/bone loss wasreversed.

The use of this invention over a twelve-week period by menopausal andpostmenopausal subjects showed a decreased level of bone resorption. Thegroup at the most risk for fractures had the greatest benefit inachieving a decrease to normal bone resorption levels. The subjects atrelatively minimal risk for fractures also benefitted with delayed boneloss. The continued use of Hexaminacol, containing bone buildingprecursors, appears to slow bone loss and shows great promise in helpingto build bone which over time will be evident on BMD measurements.

Example 6 Informal Case Study: Chapel Hill, N.C.

A group of women who were diagnosed with osteoporosis with a T-score of−2.5 or lower were prescribed an anti-resportive agent, Fosamax which isa bisphosphonate. It was recommended that they also take calciumsupplement with Vitamin D.

A group of seven women received Fosamax and Hexaminacol in place ofCalcium and Vitamin D. Another group of women were in the routinetreatment of Fosamax, calcium and Vitamin D. NTx tests were conducted ona monthly basis to monitor the progress of the treatment for threemonths for both groups. The results clearly indicated that all thepatients who were on Hexaminacol and complied with the treatment regimenbenefitted with normal NTx readings within 3 months, whereas in theCalcium supplement group only 67% of the patients showed improvements in3 months. This leads us to believe that Hexaminacol could work well inconjunction with Bishosphanates to produce good results in addressingosteoporosis issues.

Example 7 Equine Applications

In the equine athletics it is intended for bone mass building, Bonegrowth, Bone repair and maintenance of the skeletal structure. It isparticularly well suited for the racing industry where an offspringwhich is born any month of the year is considered one year old and aresubjected to training in a very young age before the full bone mass andskeletal structure is attained to withstand the rigorous trainingresulting in serious injury.

In a race track barn a 2 year old expensive colt sustained a seriouscanon bone injury resulting in a surgery where the fractured canon bonewas reattached by drilling a hole in the bottom of the fracture and topof the fractured bone and secured with a pin. The leg then wasimmobilized and the colt was administered 30-60 grams of Hexaminacol.The new compound was sprinkled on top of the grain ration twice a day of15-30 grams per treatment. The colt had no problem consuming Hexaminacoland no side reactions were noted during the entire treatment period. Atthe end of month 1 treatment there were no significant improvementsnoticed in the x-rays. The treatment continued and at the end of month 2substantial formation of bone was noticed in the x-rays. The trainer andthe veterinary surgeon felt that calcification is taking place at arapid rate. The treatment was continued and the end of 3 months therewas a complete healing of the canon bone resulting in a much strongerbone structure than the normal unbroken leg. The colt was back intraining full scale and participated in a race, placing second at the4th month. The trainer quoted, “In all my 40 years of training I havenever seen such a complete healing of the canon bone injury as I haveseen in this colt.” Further experiments concluded that this is a viableoption to provide this preparation to pregnant mares, weaned colts andfillies, young adults in training and among mature animals inmaintenance of bone, muscle and tendon tissues. This preparation is aviable option for animals facing steroid treatment and also benefit inthe process of bone loss due to steroid intake in treatments.

Example 8 Canine Applications

Hexaminacol is found to be an effective treatment option for sportsanimals, show dogs and working dogs for treatment of bone fracturehealing, in hip dysplasia and skeletal growth. It is found to be avaluable option for maintenance of bone strength and bone density.Several batches of dog biscuits were made with the objective ofproviding 1.5 to 2 grams of Hexaminacol on a daily basis. The trialanimals preferred the biscuits made with Hexaminacol and did not haveany side reactions. The trial animals maintained normal roles andfurther studies are planned in this area.

Example 9 Avian and Poultry Applications

Laying hens were fed in their daily ration of 120 mgs per 7 lbs inaverage weight birds of our compound for 3 months and found thethickness and stability of egg shells and the firmness of the egg yolkand egg white were superior to control groups who were on a normal dailyration. This leads us to believe that our compound has application inpoultry industry to maintain the blood serum calcium and other mineralnutrients to produce healthy eggs. It is also applicable to the Avianbreeding programs for captive pet and zoo industry birds and hascritical application in endangered species rejuvenation programs.

The following References are incorporated by reference herein as thoughthey are part of this Application.

REFERENCES

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What is claimed is as follows:
 1. A method of making a nutritionalcomposite material comprising the steps of a) providing whey-derivedmilk minerals in solid form; b) providing milk-derived basic protein insolution; c) exposing the milk-derived protein solution of step b. to anion exchange step to enhance the weight percent milk-derived basicproteins in the solution; d) removing sodium-based minerals and sodiumsalts from the milk-derived basic protein solution; e) purifying themilk-derived solid minerals by mixing the minerals with a solvent andheating the solution; f) combining the purified milk-derived solidmineral solution of the previous step with the enhanced milk-derivedbasic protein solution of step d); g) removing the solvent of theprevious step to produce the nutritional composite material.
 2. A methodaccording to claim 1 in which additional milk-derived protein insolution is treated according to step c. to increase the amount ofmilk-derived basic protein in solution to be treated according to stepd).
 3. A nutritional composite material made according to the process ofclaim
 1. 4. A material made according to claim 3 which has an x-raydiffraction pattern as shown in FIG.
 18. 5. A method according to claim1 wherein step d is accomplished using a dialysis step.
 6. A methodaccording to claim 1 wherein step e) is accomplished by mixing themilk-derived solid minerals with water and heating the mixture.
 7. Amethod according to claim 1 wherein steps a) and b) are accomplished bycentrifugation of a solution of whey-derived milk minerals.
 8. A methodaccording to claim 1 wherein step e) is followed by concentrating thepurified solid minerals.
 9. A method according to claim 1 wherein stepg) is accomplished by drying.